Angiotensin II (Ang II) signaling, including matrix metalloproteinase type II (MMP2) activation, has been linked to an age-associated increase in migration capacity of vascular smooth muscle cells (VSMC), and to other proinflammatory features of arterial aging. Calpain-1 activation is required for MMP2 expression in fibroblasts and is induced in cardiomyocytes by Ang II. The consequences of engagement of calpain-1 with its substrates, however, in governing the age-associated proinflammatory status within the arterial wall, remain unknown. 1. The present findings demonstrate that transcription, translation, and activity of calpain-1 are significantly up-regulated in rat aortae or early-passage aortic VSMC from old (30-mo) rats compared to young (8-mo). 2. Dual immunolabeling of the arterial wall indicates that colocalization of calpain-1 and Ang II increases within the aged arterial wall. To further explore the relationship of calpain-1 to Ang II, we chronically infused Ang II into young rats, and treated cultured aortic rings or VSMC with Ang II. We also constructed adenoviruses harboring calpain-1 (CANP1) or its endogenous inhibitor calpastatin (CAST) and infected these into VSMC. Ang II induces calpain-1 expression in the aortic walls in vivo and ex vivo and VSMC in vitro. The Ang II mediated, age-associated increased MMP2 activity and migration in VSMC are both blocked by calpain inhibitor 1 or CAST. 3. Over-expression of calpain-1 in young VSMC results in cleavage of intact vimentin, and an increased migratory capacity mimicking that of old VSMC, which is blocked by the MMP inhibitor, GM6001. Age-associated arterial alterations are characterized by both a shift of vascular smooth muscle cells (VSMC) from a contractile to synthetic phenotype, and extracellular matrix remodeling (ECM), which includes collagen over-deposition and vascular calcification (VC). Calcified ECM is a salient feature of the aged arterial wall. Secreted matrices from aged VSMC produce a favorable microenvironment of VC. We then investigate the role of calpain-1 in regulating MMP2 activation and ECM remodeling especially VC. 4. Histo-immunostaining shows that calpain-1 colocalization with MMP2 in old rat vascular smooth muscle cells (VSMC). Over-expression of calpain-1 in young VSMC induced MMP2 transcripts, protein and activity levels. Furthermore, our results show that over-expression of calpain-1 induced imbalance of MMP2 regulators (MT1MMP/TIMP2), which are the key molecules for MMP2 activation. 5. Our results also show that over-expression of calpain-1 by a recombinant adenovirus (pAd/CANP1) infection in young VSMC increased Col I by 2.18- fold and Col III by 1.99-fold compared to control virus (pAd/GFP), up to levels of old control cells, which is both the major characteristic of ECM remodeling and the alternative way to induce MMP2 activation in the aged arterial wall. 6. Interestingly, over-expression of calpain-1 in young rat VSMC facilitates VC by inducing TGF-beta1/Smad signaling, elastin degradation, collagen II expression, ALP activation and total intracellular calcium content together with the reduced VC inhibitors osteopontin (OPN) and osteonectin (ON) (Table 1). Taken together, our data strongly indicate that the increased calpain-1 activity within VSMCs plays a pivotal role by regulating MMP2 in ECM remodeling especially by enhanced collagen production and VC with central arterial aging. Table 1 8-mo CANP1 vs GFP TGFbeta1 238% Smad 2/3 242% Ela tin 56% Collagen II 38% ALP activity 35% Ca2+ content 72% OPN 65% ON 30% BMP2 No change